1. Field of the Invention
The present invention relates generally to variant red fluorescent proteins (RFPs), and more specifically to Anthozoan fluorescent proteins (AnFP), having at least one amino acid alteration that results in more efficient maturation than the corresponding wild-type protein or another variant RFP from which such variants derive. The invention further concerns RFP variants that additionally have reduced propensity tetramerize, and thus form predominantly monomeric and/or dimeric structures. The invention also relates to methods of making and using such RFP variants.
2. Description of the Related Art
The identification and isolation of fluorescent proteins in various organisms, including marine organisms, has provided a valuable tool to molecular biology. The green fluorescent protein (GFP) of the jellyfish Aequorea victoria, for example, has become a commonly used reporter molecule for examining various cellular processes, including the regulation of gene expression, the localization and interactions of cellular proteins, the pH of intracellular compartments, and the activities of enzymes.
The usefulness of Aequorea GFP has led to the identification of numerous other fluorescent proteins in an effort to obtain proteins having different useful fluorescence characteristics. In addition, spectral variants of Aequorea GFP have been engineered, thus providing proteins that are excited or fluoresce at different wavelengths, for different periods of time, and under different conditions. The identification and cloning of a red fluorescent protein from Discosoma coral, termed DsRed or drFP583, has raised a great deal of interest due to its ability to fluoresce at red wavelengths.
The DsRed from Discosoma (Matz et al., Nature Biotechnology 17:969-973 [1999]) holds great promise for biotechnology and cell biology as a spectrally distinct companion or substitute for the green fluorescent protein (GFP) from the Aequorea jellyfish (Tsien, Ann. Rev. Biochem., 67:509-544[1998]). GFP and its blue, cyan, and yellow variants have found widespread use as genetically encoded indicators for tracking gene expression and protein localization and as donor/acceptor pairs for fluorescence resonance energy transfer (FRET). Extending the spectrum of available colors to red wavelengths would provide a distinct new label for multicolor tracking of fusion proteins and together with GFP (or a suitable variant) would provide a new FRET donor/acceptor pair that should be superior to the currently preferred cyan/yellow pair (Mizuno et al., Biochemistry 40:2502-2510 [2001]).
One problem associated with the use of DsRed as a fluorescent report is its slow and inefficient chromophore maturation. Most previous attempts to improve the rate and/or extent of maturation of DsRed (Verkhusha et al., J. Biol. Chem., 276:29621-29624 [2001]; and Terskikh et al., J. Biol. Chem., 277:7633-7636 [2002]) including the commercially available DsRed2 (CLONTECH, Palo Alto, Calif.), have provided only modest improvements. Recently, an engineered variant of DsRed, known as T1(shown in FIG. 1A), has become available and effectively solved the problem of the slow maturation (Bevis and Glick, Nat. Biotechnol., 20:83-87 [2002]). However this variant appears to still suffer from an incomplete maturation and therefore like DsRed, a significant fraction of the protein remains as the green fluorescent intermediate in the aged tetramer.
Furthermore, all coelenterate fluorescent proteins cloned to date display some form of quaternary structure, including the weak tendency of Aequorea green fluorescent protein (GFP) to dimerize, the obligate dimerization of Renilla GFP, and the obligate tetramerization of the Discosoma DsRed (Baird et al., Proc. Natl. Acad. Sci. USA 97:11984-11989 [2000]; and Vrzheshch et al., FEBS Lett., 487:203-208 [2000]). While the weak dimerization of Aequorea GFP has not impeded its acceptance as an indispensable tool of cell biology, the obligate tetramerization of DsRed has greatly hindered its development from a scientific curiosity to a generally applicable and robust tool, most notably as genetically encoded fusion tag.
DsRed tetramerization presents an obstacle for the researcher who wishes to image the subcellular localization of a red fluorescent chimera, as the question exists as to what extent will fusing tetrameric DsRed to the protein of interest affect the location and function of the latter. Furthermore, it can be difficult in some cases to confirm whether a result is due, for example, to a specific interaction of two proteins under investigation, or whether a perceived interaction is an artifact caused by the oligomerization of fluorescent proteins linked to each of the two proteins under investigation. There have been several published reports (see, e.g., Mizuno et al., Biochemistry 40:2502-2510 [2001]; and Lauf et al., FEBS Lett., 498:11-15 [2001]) and many unpublished anecdotal communications, in which DsRed chimeras have been described as forming intracellular aggregates that have lost their biological activity. DsRed also suffers from slow and incomplete maturation (Baird et al., Proc. Natl. Acad. Sci. USA 97:11984-11989 [2000]).
One approach to overcome these shortcomings has been to continue the search for DsRed homologues in sea coral and anemon; an approach that has yielded several red shifted proteins (Fradkov et al., FEBS Lett., 479:127-130 [2000]; and Lukyanov et al., J. Biol. Chem., 275:25879-25882 [2000]). However, the fundamental problem of tetramerization has yet to be overcome. The only published progress towards decreasing the oligomeric state of a red fluorescent protein involved an engineered DsRed homologue, commercially available as HcRed1 (CLONTECH), which was converted to a dimer with a single interface mutation (Gurskaya et al., FEBS Lett., 507:16-20 [2001]). Although HcRed1 has the additional benefit of being 35 nm red-shifted from DsRed, it is limited by a low extinction coefficient (20,000 M-1cm-1) and quantum yield (0.015) (CLONTECH Laboratories Inc., (2002) Living Colors User Manual Vol. II: Red Fluorescent Protein [Becton, Dickinson and Company], p. 4) making the protein problematic to use in experimental systems.
A methionine is found at position 66 of a tetrameric nonfluorescent chromoprotein from Anemonia sulcata which was converted to a fluorescent protein through the introduction of two mutations (Lukyanov, K. A., Fradkov, A. F., Gurskaya, N. G., Matz, M. V., Labas, Y. A., Savitsky, A. P., Markelov, M. L., Zaraisky, A. G., Zhao, X., Fang, Y. et al. (2000) J. Biol. Chem. 275, 25879-25882). Introduction of a methionine at position 66 apparently improves the fluorescent properties of both a green (zFP506) and a cyan (amFP486) tetrameric fluorescent protein though no details have been published (Yanushevich, Y. G., Staroverov, D. B., Savitsky, A. P., Fradkov, A. F., Gurskaya, N. G., Bulina, M. E., Lukyanov, K. A. & Lukyanov, S. A. (2002) FEBS Lett. 511, 11-14).
Thus, there exists a need in the art for the development of red fluorescent polypeptides that find use in scientific applications without technical limitations due to inefficient and slow chromophore maturation, and oligomerization, especially tetramerization. There exists a need for methods to produce red fluorescent proteins (RFPs) exhibiting more efficient chromophore maturation than wild-type RFPs or other RPF variants. Furthermore, there exists a need for RFPs that additionally have reduced propensity for oligomerization, such as, tetramerization. Most significantly, there exists a need for RFP variants with improved efficiency of maturation that demonstrate useful fluorescence in a monomeric state in experimental systems. The present invention satisfies these needs and provides additional advantages.